In higher vertebrates, the luminal surface of the small intestine consists of a specialized epithelium adapted to enhance nutrient absorption through the presence of numerous villi, finger-like projections that dramatically increase surface area in the gut. Cells of the villus have a lifetime of only 3 to 4 days, after which they are replaced by cells originating at the villus base in crypts, stem cell niches within invaginations of the epithelium. Wnt signaling plays an important role in regulating the differentiation and proliferation of these stem cells, and abnormal Wnt-signaling in these structures has been strongly associated with gastrointestinal cancers. At the onset of villi morphogenesis, the gut tube lumen is initially smooth, and the inner endodermal layer represents a uniform pool of epithelial stem cells. Villi morphogenesis proceeds through striking transformations in luminal topography, whereby a number of straight longitudinal ridges form, then become wavy, or kinked, and finally villi begin to form at the points of inflection in these ridges. By the end of this transformation, stem cell populations have become restricted to intervillus regions where the crypts will form. These transformations generate physical patterns consistent with compression induced bi-directional buckling of an elastic material, suggesting that mechanical forces may plan an important role in villi morphogenesis. Therefore, the objective of the proposed work is to determine the physical forces that drive topographical changes of the midgut endoderm, and what role this unique topography plays in patterning the intestinal villi. It is hypothesized that physical forces generated in the surrounding mesodermal layers drive compression- induced buckling in the endoderm, and that this displaces endodermal cells within a morphogen gradient, providing positional cues that guide differentiation into mature villi. The following aims are proposed: Specific Aim 1: Determine the role of active force generation in the formation and kinking of pre-villus longitudinal ridges. Specific Aim 2: Determine the expression pattern of Wnts and related genes in the midgut mesoderm and endoderm throughout the stages of villus morphogenesis. Specific Aim 3: Determine the dependence of villus morphogenesis on endodermal topography. These investigations will be carried out in the chick, a model system that closely resembles human gastrointestinal development, and will rely on a combination of mechanically motivated physical manipulations and genetic ones to identify how developing tissues may exploit physical forces to position cells locally within spatially varying signaling gradients in order to specify their adult form. The proposed work may not only advance our knowledge of how gastrointestinal cancers form and can be treated, but may aid in understanding the fundamental mechanisms by which vertebrate morphogenesis proceeds through an integration of mechanical, molecular, and positional cues. PUBLIC HEALTH RELEVANCE: In humans and other higher vertebrates, the cells that form the lining of the adult intestine are organized into many finger-like projections, termed villi, that are essential for nutrient absorption. These cells live for only days before they are replaced by new cells that originate in a special compartment of stem cells, known as crypts. Unfortunately, if these villi do not form normally during embryonic development, the intestine cannot perform its function, and if during adulthood, the signals that sustain this cycle of stem cell differentiation become perturbed, lethal cancers can form in the small intestine and colon. Because during embryonic development, the cells that create these crypts and villi are initially stem-cell like, understanding how the villi form, and how stem cells become regulated and restricted to the crypts during development will help determine how gastrointestinal cancers form and may reveal targets for their treatment. The objective of the proposed work is to determine the physical forces and molecular pathways responsible for the development of intestinal villi.